Molecular Cloning and Chromosomal Localization of Human Hippocalcin Gene.

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Hippocalcin is a 23 kDa calcium-binding protein with a three EF-hand structure, primarily idenditified in the rat Hippocampus. Hippocalcin has a sequence homology with recoverin, suggesting that hippocalcin may regulate phosphorylation of receptor molecules in the hippocampus. To investigate a possible relationship of hippocalcin to human neurological disorders, we have analyzed the human hippocalcin gene. A human Hippocampus cDNA library was screened with a rat hippocalcin cDNA probe. Eight independent cDNA clones were obtained and classified into five species (hHLP1-5) based on the restriction enzyme maps. The clone hHLP1 encode the entire open reading frame of human hippocalin. The predicted amino acid sequence was 100% homologous to that of rat hippocalcin. After screening a human genomic DNA cosmid libray, we obtained 3 independent clones. All these 3 clones exhibited similar restriction fragment patterns. The human hippocalcin gene spans approximately 15-kb and contains 3 exons. The exon sequence of the human hippocalcin gene perfectly matched its cDNA sequence, and all exon-intron junctions conformed to the GT/AG rule. The intron positions are not consistently placed with respect to the coding regions of the tandemly repeated EF-hand domains. Southern blot analysis of the human genomic DNAs and cosmid clones with a human hippocalcin cDNA probe indicated that the human hippocalcin gene is present as a single copy gene in the haploid genome. The transcription initiation site was determined by primer extension assay with poly (A) + RNA from human brain and was assigned to the adenine residue at the position -181. In 2-kb sequence upstream of the transcription initiation site, no common motifs for mammalian promoters such as TATA-, GC- and CAAT-boxes appear to be present. By direct R-banding fluorescence in situ hybridization, the human hippocalcin gene was assigned to bands 1p34.2-35.